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Aim: Lyme borreliosis (LB) is the most common tick-borne disease in Germany; however, data on the economic burden of LB are limited. In this study, we aim to report healthcare costs, healthcare resource utilisation (HCRU) and diagnostic consumption associated with LB by clinical manifestation., Method: Using specific case definitions, patients with localised disease (erythema migrans [EM]) or disseminated disease (Lyme arthritis [LA], Lyme neuroborreliosis [LNB] and other rarer manifestations [OTH]) were identified from a claims database in 2016 and followed up for 3 years (2016-2019). After propensity score matching, excess costs and HCRU were calculated as the differences between each LB cohort and the matched control cohort., Results: On a per-patient basis, the excess all-cause healthcare cost was €130 for EM during Quarter 1 of Year 1, and €1539 for LA, €3248 for LNB and €4137 for OTH during Year 1. Only for OTH, additional €1860 was observed in Year 2. No increase in costs was observed in Year 3. When extrapolated to all German patients with statutory health insurance, LB was associated with €64.5 million in excess costs. Although disseminated manifestations only accounted for 7.8% of all LB cases, they were responsible for 66% of overall costs. In addition, LB patients consumed healthcare resources of 1.4 million excess outpatient visits, 13,000 excess hospitalisations, 96,000 ELISAs and 65,000 Western blots., Conclusion: This study shows the substantial economic burden of LB to the German healthcare system., (© 2024 Pfizer Pharma GmbH. Zoonoses and Public Health published by Wiley‐VCH GmbH.)

Introduction: Lyme borreliosis (LB), an infection caused by Borrelia burgdorferi sensu lato (Bbsl), is the most common tick-borne disease in Europe. To further characterize the LB burden in the Czech Republic, we conducted a seroprevalence study and estimated the incidence of symptomatic Bbsl infections., Methods: Anti-Bbsl IgM and IgG antibodies were detected in sera collected from the adult population in 2011 -2012 by enzyme-linked immunosorbent assay and immunoblot tests at the National Reference Laboratory. The incidence of symptomatic Bbsl infections was estimated from the seroprevalence results and the symptomatic proportion and duration of persistence of anti-Bbsl IgG antibodies in Bbsl-infected individuals. Surveillance under-detection of symptomatic Bbsl infections was estimated by comparing surveillance-reported and seroprevalence-based incidence., Results: Samples from 1996 adults were tested; the median age (range) was 45 (18 -87) years; 1037 (52.0 %) were female. The prevalence (with 95 % confidence interval) of anti-Bbsl IgG, and IgM and/or IgG (IgM/IgG) antibodies was 6.3 % (5.3 -7.5 %), and 9.5 % (8.3 -10.9 %), respectively. The IgM/IgG prevalence was 7.8 % (6.5 -9.2 %) in Bohemia and 15.3 % (12.2 -19.0 %) in Moravia. There were an estimated 30,563 (26,550 -34,962) symptomatic incident Bbsl infections in adults in the Czech Republic in 2012, for an incidence of 352.2 (306.0 -402.9) symptomatic Bbsl infections per 100,000 adults per year. There were an estimated 11 (10 -13) symptomatic Bbsl infections for each surveillance-reported LB case in the Czech Republic in 2012., Conclusions: There is high incidence of symptomatic Bbsl infections in the Czech Republic, particularly in Moravia. Interventions are needed to address the substantial burden of LB in the Czech Republic., (Copyright © 2025 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Introduction: Health claims data are a valuable resource for health services research, enabling analysis of the costs of hospitalizations, outpatient visits, procedures, and medications, and providing an improved understanding of the economic burden and underlying cost drivers for a given health condition. Since no recent data were available from Germany on the medical costs and clinical outcomes of Clostridioides difficile infections (CDI), this study assessed the economic burden of CDI and all-cause mortality in adults in Germany., Methods: A retrospective cohort study was conducted using a large, anonymized administrative health claims research database from Germany from which an age- and sex-representative sample of 4 million insured persons covered by approximately 60 statutory health insurances was extracted. Propensity score matching was conducted on age, sex, comorbidities, and antibiotic use to identify four matched controls (i.e., patients without CDI) for every eligible adult patient with CDI (i.e., case) in the study cohort. Costs, healthcare resource utilization, and CDI-attributable all-cause mortality were assessed., Results: Overall, there were 15,342 CDI cases in the study cohort. One-year mortality in CDI cases (45.7%) was more than fourfold that of matched non-CDI controls (11.0%). In the year following the index date, average mortality-adjusted medical costs per person-time for CDI cases were almost fivefold that of matched non-CDI controls, representing a cost difference of €31,459, mainly driven by inpatient treatment. Overall excess costs for CDI cases were estimated at approximately €1.6 billion within 1 year after diagnosis., Conclusions: CDI in Germany is associated with a high clinical and economic burden, including significantly higher mortality, costs, and healthcare resource utilization, in patients with CDI versus their matched patients without CDI. This has important implications for patients, healthcare providers, and the healthcare system., Competing Interests: Declarations. Conflict of interest: Katharina Schley, Gordon Brestrich, and Christof von Eiff are employees of Pfizer Pharma GmbH; Kirstin Heinrich is an employee of Pfizer Inc; and Jennifer C. Moïsi is an employee of Pfizer Vaccines—all of whom may hold stock or stock options in Pfizer Inc. Dennis Häckl is an employee of WIG2, which received funding from Pfizer in connection with the development of this manuscript. Dominik Obermüller is an employee of InGef, which acted as subcontractor and received funding from WIG2 in connection with the development of this manuscript. Thomas Weinke received an honorarium from Pfizer in connection with the development of the study. Ethical approval: Additional ethics approval was not required as the data were anonymized, and the study, which was a retrospective analysis of an anonymized health claims research database, complied with German data protection regulations and federal law., (© 2024. Pfizer, Inc.)

Objectives: To better understand the Lyme borreliosis (LB) burden in Europe, we aimed to estimate the incidence of symptomatic Borrelia burgdorferi sensu lato (Bbsl) infections after adjusting public health LB surveillance data for under-detection of symptomatic Bbsl infections., Methods: Data from seroprevalence studies and estimates of the symptomatic proportion and duration of antibody detection in Bbsl-infected individuals, derived from reviews of the published literature, were used to adjust public health LB surveillance data to estimate the incidence of symptomatic Bbsl infection in nine European countries from 2018 to 2022., Results: The prevalence of anti-Bbsl antibodies ranged from 2.3% in Romania to 9.4% in Germany. Under-detection multipliers varied across surveillance systems; using 10-year duration of antibody detection, multipliers were 2.4-10.5 in countries reporting all LB cases and 54.6-722.2 in countries reporting only Lyme neuroborreliosis cases. The incidence of symptomatic Bbsl infection adjusted for under-detection was highest in Finland, Germany, Norway, Poland, and Switzerland, intermediate in the Czech Republic and Denmark, and lowest in Ireland and Romania., Conclusion: Adjustment of LB surveillance for under-detection found a high incidence of symptomatic Bbsl infection in several European countries. Differences in LB surveillance systems should be considered when comparing surveillance data between countries and when estimating LB disease burden., Competing Interests: Declarations of competing interest Frederick J. Angulo, Gordon Brestrich, Emily Colby, Andrew Vyse, L. Hannah Gould, Kate Halsby, Patrick H. Kelly, Jennifer C. Moïsi, Andreas Pilz, Madiha Shafquat, and James H. Stark are employees of Pfizer and may hold stock or stock options. Per-Eric Lindgren has been an external scientific advisor to Valneva, Pfizer, and Bavarian-Nordic A/S., (Copyright © 2024 Pfizer Inc. Published by Elsevier Ltd.. All rights reserved.)

Lyme borreliosis (LB), the most common tick-borne disease in Europe, is endemic to southern coastal Norway. LB commonly presents as erythema migrans, which can disseminate, resulting in more severe disease such as Lyme neuroborreliosis or arthritis. In Norway, public health LB surveillance is conducted via mandatory reporting of laboratory-confirmed disseminated cases. From 2012 to 2022, Norway's surveillance-reported incidence of laboratory-confirmed disseminated LB increased by 78%. Although surveillance provides estimates of the incidence of disseminated LB, this study sought to estimate the incidence of symptomatic LB to better understand Norway's LB disease burden. Two studies were identified that, when combined, estimated an LB seroprevalence of 6.8% in the general adult population in southern Norway. Utilizing data from these seroprevalence studies, public health surveillance, and results from literature searches indicating that 37% of seroconverted LB cases are symptomatic and that the duration of LB antibody detection ranges from 10 to 20 years, we estimated that there were 315-630 symptomatic LB cases per 100,000 adult population in five southern coastal counties in Norway in 2022 and 24-48 cases of symptomatic LB for every public health surveillance-reported LB case in adults in these five counties in Norway., (© 2024 The Author(s). APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Background: Respiratory syncytial virus (RSV) is a common cause for severe lower respiratory tract infections (LRTI) in children < 2 years of age in Germany - though little is known about the clinical and economic burden of RSV in children with and without risk factors per month of life., Methods: In a retrospective health claims analysis, we identified RSV inpatient cases between 2014 and 2019. We assessed incidence rates, mortality rate, healthcare resource utilization, associated direct costs per case and excess costs for 30, 90 and 365 days after hospital admission matched to a control group. The outcomes are reported separately for the first and second year of life (i.e., for infants and toddlers) and were stratified by month of life, preterm and risk status (i.e., presence of underlying disease: chronic respiratory or cardiac disease, immunosuppression, neurological diseases, diabetes, conditions originating in the perinatal period)., Results: RSV-attributable hospital incidence rate was higher in infants (30.25/1,000) than toddlers (14.52/1,000), highest in the first three months of life (44.21/1,000), in infants born preterm (64.76/1,000) or with any underlying disease (54.85/1,000). Mortality rate was also higher for infants (0.08/1,000) than toddlers (0.04/1,000). Mean 30-day excess costs ranged from 2,953 € for infants born full-term at no risk, hospitalized for 5 days, to 6,694 € for infants born extremely premature, hospitalized for 7 days., Conclusion: In Germany, the clinical and economic burden of RSV is substantial, especially in the most vulnerable population, that is, very young infants, those born premature and/or those with an underlying disease., (© 2024. The Author(s).)

Background: Lyme borreliosis (LB), caused by Borrelia burgdorferi sensu lato (Bbsl), is the most common tick-borne disease in Europe. Although public health surveillance for LB has been conducted in Romania since 2007, the extent of under-detection of Bbsl infections by LB surveillance has not been estimated. We therefore estimated the under-detection of symptomatic Bbsl infections by LB surveillance to better understand the LB burden in Romania., Methods: The number of incident symptomatic Bbsl infections were estimated from a seroprevalence study conducted in six counties (population 2.3 M) and estimates of the symptomatic proportion and duration of persistence of anti-Bbsl immunoglobulin G (IgG) antibodies. The number of incident symptomatic Bbsl infections were compared with the number of surveillance-reported LB cases to derive an under-detection multiplier, and then the under-detection multiplier was applied to LB surveillance data to estimate the incidence of symptomatic Bbsl infection from 2018 to 2023., Results: We estimate that there were 1968 individuals with incident symptomatic Bbsl infection in the six counties where the seroprevalence study was conducted in 2020, compared with the 187 surveillance-reported LB cases, resulting in an under-detection multiplier of 10.5 (i.e., for every surveillance-reported LB case, there were 10.5 symptomatic incident Bbsl infections). The incidence of symptomatic Bbsl infection in the six counties was 86.9/100,000 population in 2023, similar to the incidence in 2018-2020 (86.0) and higher than in 2021-2022 (40.3)., Conclusions: There is a higher incidence of symptomatic Bbsl infection than is reported through public health surveillance for LB in Romania. Additional efforts are needed to strengthen disease prevention and address the important public health problem of LB., (© 2024. The Author(s).)

Lyme borreliosis (LB) is the most common tick-borne disease in Germany. Although the incidence of LB in Germany has been assessed in several studies, those studies either used data from statutory surveillance, which frequently underreport cases, or data from health claims databases, which may overestimate cases due to non-specific LB case definitions. Here, using a more specific case definition, we describe the incidence of medically-attended LB by disease manifestation, age group, and federal state for the period 2015-2019. Both inpatient and outpatient cases were analyzed from a claims database. To be eligible for inclusion, patients were required to have an LB specific ICD-10 GM diagnosis code plus an antibiotic prescription, and for disseminated manifestations, a laboratory test order additionally. LB cases were classified as erythema migrans (EM), or disseminated disease including Lyme arthritis (LA), Lyme neuroborreliosis (LNB), and all other disease manifestations (OTH). Between 2015 and 2019, the incidence of medically-attended LB cases ranged from 195.7/100,000 population per year (95% confidence interval [CI], 191.0 - 200.5) to 254.5/100,000 population per year (95% CI, 249.0 - 260.0) per year. The majority of cases (92.2%) were EM, while 2.8% presented as LA, 3.8% as LNB, and 1.2% as OTH. For both EM and disseminated disease, the incidence peaked in children aged 5-9 years and in older adults. By federal state, the incidence of medically-attended EM ranged from 74.4/100,000 population per year (95% CI, 71.9 - 77.0) per year in Hamburg, to 394.1/100,000 population per year (95% CI, 370.7 - 417.6) per year in Saxony, whereas for medically-attended disseminated disease, the highest incidence was in Thuringia, Saxony, and Bavaria (range: 22.0 [95% CI, 19.9 - 24.0] to 35.7 [95% CI, 34.7 - 36.7] per 100,000 population per year). This study comprehensively estimated the incidence of all manifestations of medically-attended LB and showed a high incidence of LB throughout Germany. Results from the study support performing epidemiological studies in all federal states to measure the burden of LB and to invest in public health interventions for prevention., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gordon Brestrich reports financial support and writing assistance were provided by Pfizer Pharma GmbH, Berlin, Germany. Gordon Brestrich reports a relationship with Pfizer Pharma GmbH, Berlin, Germany that includes: employment. This study was supported and jointly funded by Valneva and Pfizer as part of their co–development of a Lyme disease vaccine. All authors had complete autonomy for the process of designing the study, carrying out the analysis, interpreting the results and writing the manuscript. GB, AP, JHS, FJA, and HY are employees of Pfizer in Germany, Austria and the US. CH is a former employee of Pfizer in Germany and was involved in the design of this study, developing the study protocol, and in analyzing the data. JD and NK are employees of WIG2 GmbH, which is an independent insti-tute and paid consultant to Pfizer Pharma GmbH for designing the study, carrying out the analysis, interpreting the results and writing the manuscript. JS received an honorarium from Pfizer Pharma GmbH in connection with designing the study and interpreting the results. JS received not further funding from another company or other partners. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Introduction: Respiratory syncytial virus (RSV) burden in adults is underestimated mainly due to unspecific symptoms and limited standard-of-care testing. We estimated the population-based incidence of hospitalization and mortality attributable to RSV among adults with and without risk factors in Germany., Methods: Weekly counts of hospitalizations and deaths for respiratory, cardiovascular, and cardiorespiratory diseases were obtained (Statutory Health Insurance database, 2015-2019). A quasi-Poisson regression model was fitted to estimate the number of hospitalizations and deaths attributable to RSV as a function of periodic and aperiodic time trends, and viral activity while allowing for potential overdispersion. Weekly counts of RSV and influenza hospitalizations in children < 2 years and adults ≥ 60 years, respectively, were used as viral activity indicators. Models were stratified by age group and risk status (defined as presence of selected comorbidities)., Results: Population-based RSV-attributable hospitalization incidence rates were high among adults ≥ 60 years: respiratory hospitalizations (236-363 per 100,000 person-years) and cardiorespiratory hospitalizations (584-912 per 100,000 person-years). RSV accounted for 2-3% of all cardiorespiratory hospitalizations in this age group. The increase in cardiorespiratory hospitalization risk associated with underlying risk factors was greater in 18-44 year old persons (five to sixfold higher) than in ≥ 75 year old persons (two to threefold higher)., Conclusions: This is a first model-based study to comprehensively assess adult RSV burden in Germany. Estimated cardiorespiratory RSV hospitalization rates increased with age and were substantially higher in people with risk factors compared to those without risk factors. Our study indicates that RSV, like other respiratory viruses, contributes to both respiratory and cardiovascular hospitalizations. Effective prevention strategies are needed, especially among older adults ≥ 60 years and among adults with underlying risk factors., (© 2024. Pfizer.)

Introduction: Clostridioides difficile infection (CDI) is increasingly recognized as a public health threat at the community level in addition to being one of the most common causes of healthcare-associated infections. In Germany, the epidemiology of CDI is primarily informed by national hospital-based CDI surveillance. We used health claims data from Germany to obtain valuable insights on population-level disease burden and risk factors for CDI., Methods: This was a retrospective cohort study using a representative sample from the InGef research database. Overall and age- and sex-stratified CDI incidence rates were estimated for German adults from 2013 to 2017 using different case definitions (i.e., main, broad, strict), and further stratified by setting (inpatient versus outpatient). Risk factors for CDI were assessed for the 2013-2016 period., Results: The CDI incidence rate was high but declined by 15.3% from 2013 [141 (95% confidence interval, CI 137-145) cases/100,000 person-years] to 2017 [120 (95% CI 116-123)]. Annual CDI incidence rates were higher in female patients and the elderly. The most important risk factors for CDI were chronic inflammatory bowel disease [odds ratio (OR) 4.7, 95% CI 4.0-5.5], chemotherapy (OR 4.7, 95% CI 4.1-5.2), chronic kidney disease (OR 2.9, 95% CI 2.6-3.3), and ciprofloxacin receipt (OR 2.6, 95% CI 2.4-2.8)., Conclusions: Despite prevention strategies leading to declining incidence, CDI remains an important public health threat in Germany, with a high burden in the hospital setting and an outpatient epidemiology that is poorly understood. These findings, which are relevant both regionally and globally, can be used as a basis for further research on the full burden of CDI in Germany., (© 2023. The Author(s).)

Background: Public surveillance of Lyme borreliosis (LB) occurs in 9 out of 16 federal states of Germany and remains a critical facet of disease epidemiology and trends. We describe the incidence, time trends, seasonality, and geographic distribution of LB in Germany using publicly reported surveillance data. Methods: We obtained LB cases and incidence (2016-2020) from the online platform SurvStat@RKI 2.0, maintained by the Robert Koch Institute (RKI). Data included clinically diagnosed and laboratory-confirmed LB reported by nine out of 16 federal states of Germany where LB notification is mandatory. Results: During 2016-2020, the nine federal states reported 63,940 LB cases, of which 60,570 (94.7%) were clinically diagnosed, and 3370 (5.3%) also had laboratory confirmation, with an average of 12,789 cases annually. Incidence rates were mostly stable over time. The average annual LB incidence was 37.2/100,000 person-years and varied by spatial level, ranging from 22.9 to 64.6/100,000 person-years among nine states; from 16.8 to 85.6/100,000 person-years among 19 regions; and from 2.9 to 172.8/100,000 person-years among 158 counties. Incidence was lowest among persons 20-24 years old (16.1/100,000 person-years) and highest among those 65-69 years old (60.9/100,000 person-years). Most cases were reported between June and September, with a peak in July of every year. Conclusion: The risk of LB varied substantially at the smallest geographic unit and by age group. Our results underscore the importance of presenting LB data at the most spatially granular unit and by age to allow implementation of efficient preventive interventions and reduction strategies.

Introduction: Clostridioides difficile infection (CDI) is a recognized global threat especially for vulnerable populations. It is of particular concern to healthcare providers as it is found in both hospital and community settings, with severe courses, frequent recurrence, high mortality and substantial financial impact on the healthcare system. The CDI burden in Germany has been described and compared by analysing data from four different public databases., Methods: Data on hospital burden of CDI have been extracted, compared, and discussed from four public databases for the years 2010-2019. Hospital days due to CDI were compared to established vaccine preventable diseases, such as influenza and herpes zoster, and also to CDI hospitalisations in the United States (US)., Results: All four databases reported comparable incidences and trends. Beginning in 2010, population-based hospitalised CDI incidence increased to a peak of > 137/100,000 in 2013. Then, incidence declined to 81/100,000 in 2019. Hospitalised patients with CDI were predominantly > 50 years of age. The population-based incidence of severe CDI was between 1.4 and 8.4/100,000 per year. Recurrence rates were between 5.9 to 6.5%. More than 1,000 CDI deaths occurred each year, with a peak of 2,666 deaths in 2015. Cumulative CDI patient days (PD) were between 204,596 and 355,466 each year, which exceeded cumulated PD for influenza and herpes zoster in most years, though year-to-year differences were observed. Finally, hospitalized CDI incidence was higher in Germany than in the US, where the disease is well recognized as a public health threat., Conclusions: All four public sources documented a decline in CDI cases since 2013, but the disease burden remains substantial and warrants continued attention as a severe public health challenge., (© 2023. The Author(s).)

Clostridioides difficile infection (CDI) often manifests as diarrhea, particularly in adults of older age or with underlying comorbidities. However, only severe cases are notifiable in Germany. Moreover, failure to collect a stool specimen from inpatients with diarrhea or incomplete testing may lead to underdiagnosis and underreporting of CDI. We assessed the frequency of diarrhea, stool specimen collection, and CDI testing to estimate CDI underdiagnosis and underreporting among hospitalized adults. In a ten-day point-prevalence study (2019-2021) of nine hospitals in a defined area (Muenster/Coesfeld, North Rhine-Westphalia, Germany), all diarrhea cases (≥ 3 loose stools in 24 h) among adult inpatients were captured via medical record screening and nurse interviews. Patient characteristics, symptom onset, putative origin, antibiotic consumption, and diagnostic stool sampling were collected in a case report form (CRF). Diagnostic results were retrieved from the respective hospital laboratories. Among 6998 patients screened, 476 (7%) diarrhea patients were identified, yielding a hospital-based incidence of 201 cases per 10,000 patient-days. Of the diarrheal patients, 186 (39%) had a stool sample collected, of which 160 (86%) were tested for CDI, meaning that the overall CDI testing rate among diarrhea patients was 34%. Toxigenic C. difficile was detected in 18 (11%) of the tested samples. The frequency of stool specimen collection and CDI testing among hospitalized diarrhea patients was suboptimal. Thus, CDI incidence in Germany is likely underestimated. To assess the complete burden of CDI in German hospitals, further investigations are needed., (© 2022. The Author(s).)

Effector memory T cells are effective in controlling acute infections, but central memory T cells play a key role in long-lasting protection against viruses and tumors. In vivo/in vitro challenge by Ag commonly supports the generation of effector memory T cells with limited longevity. To our knowledge, this study demonstrates for the first time in the human system and under rechallenge conditions that targeting IL-2R by partial mammalian target of rapamycin inhibition or blocking IL-2Rα enriches human CD4(+)/CD8(+) central memory T cells within the virus-specific T cell product associated with enhanced functionality (i.e., multicytokine secretors, including IL-2; enhanced CD137 and CD107a expression on CD8(+) and CD4(+) T cells, respectively; and killing infected target cells). Remarkably, the effects on CD8(+) T cells are mainly mediated via the enhancement of CD4(+) T cell function. The data reveal new insights into the role of CD4(+) T cell support for the quality of CD8(+) T cell memory, even under rechallenge conditions. Moreover, our method offers a new approach to improve the long-lasting efficacy of adoptive T cell therapy in patients.

Chronically immunosuppressed patients, like solid-organ-transplant (SOT) recipients, are at increased risk for severe human cytomegalovirus (HCMV) infection. Despite the availability of effective antiviral drugs, lasting control of remaining viruses is dependent on an effective T-cell immunity. So in some patients conventional antiviral therapy cannot control the infection and prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Selective reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells derived from healthy donors is a safe and effective approach in hematopoietic stem cell transplant recipients. The aim of this study was to determine whether functional HCMV-specific T cells can also be generated from chronically immunosuppressed patients. Autologous CD4+/8+ T-cell lines directed against the HCMV protein IE-1 were generated from the peripheral blood of SOT patients using a recently developed modular protocol easily applicable to good-manufacturing-practice conditions. T-cell lines from SOT patients showed similar features as cells from healthy donors regarding phenotype, functionality (HCMV-specific killing, gene expression pattern, and cytokine secretion), IE-1 epitope recognition, and dominance of distinct T-cell receptor V beta families. Most importantly, this protocol also allowed the generation of T-cell lines from immunosuppressed patients with HCMV infection/chronic HCMV disease. Our data suggest the potential of this autologous approach for the treatment of SOT recipients suffering from HCMV infection/disease poorly responding to virostatic therapy.

Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy.

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity., Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mug nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amino-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFNgamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFNgamma, IL2, IL27 and T-bet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low., Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes.

Adoptive immunotherapy with in vitro generated Epstein-Barr virus (EBV)-specific T cells is a safe and effective treatment in patients with EBV-related complications after transplantation. More frequent use of EBV-specific T cells is held back by their cost and time-intensive generation under good manufacturing practice (GMP) conditions. Currently, EBV-specific T cells are produced by repetitive stimulation of peripheral blood mononuclear cells with EBV-infected lymphoblastoid cell lines (LCLs), a protocol that requires several open GMP-handling steps. The aim of the present study was to improve T-cell generation under GMP conditions. We introduce a novel generation protocol that replaces repetitive with short-term LCL stimulation of PMBCs. Vital and formalin-fixed LCLs were used to further increase biosafety. Stimulated T cells were selected by the clinically approved cytokine secretion assay followed by nonspecific expansion. Sufficient numbers of EBV-specific T-cell lines were generated with all protocols. Specific recognition and killing of EBV-infected targets was found and was independent of the generation protocol applied. The novel protocol based on formalin-fixed cells, selection, and expansion reduced open GMP-handling steps and increased biosafety. Furthermore, fixation will allow the use of transgenic LCLs (eg, with cytomegalovirus or tumor antigens) and thereby facilitate the generation of antigen-specific T cells directed against pathogens other than EBV.

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793).